Bubr1

HeLa-FlpIn-TRex cells were induced with doxycyclin (2 µg ml⁻¹) 48 h before harvesting. Synchronization by thymidine (2 mM) for 24 h and release for 10 h into Taxol (2 µM) arrested cells in prometaphase. Cells were collected by mitotic shake-off. Lysis was done in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% NP40, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma). Complexes were purified using GFP-Trap beads (ChromoTek) for 15 min at 4°C. Precipitated proteins were washed with lysis buffer and eluted in 5× SDS sample buffer. Primary antibodies were used at the following dilutions for western blotting: BUBR1 (A300-386A, Bethyl) 1 : 2000, alpha-tubulin (T9026, Sigma) 1 : 5000, GFP (Custom) 1 : 10 000, APC1 (A301-653A, Bethyl) 1 : 2500, APC3 (gift from Phil Hieter) 1 : 2000, MAD2 (Custom) 1 : 2000, CDC20 (A301-180A, Bethyl) 1 : 1000. Western blot signals were detected by chemiluminescence using an ImageQuant LAS 4000 (GE Healthcare) imager.

Immunoblotting was performed as previously described [42 (link)], except that a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA) was used to detect the signals. The following antibodies were obtained from the indicated sources and used at the indicated concentrations: monoclonal antibodies against beta-actin (Sigma-Aldrich; 0.2 µg/ml), FLAG (Sigma-Aldrich; 1 µg/ml), BUB3 (BD Biosciences, Franklin Lakes, NJ, USA; 0.25 µg/ml), CDC27 (BD Biosciences; 0.125 µg/ml), cleaved PARP1(Asp214) (BD Biosciences; 0.2 µg/ml), CDH1 (Thermo Scientific; 1 µg/ml), CDC20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 0.2 µg/ml), MAD1 (Santa Cruz Biotechnology; 0.2 µg/ml),TRIP13 (Santa Cruz Biotechnology; 0.2 µg/ml), APC4 (Abcam, Cambridge, UK; 0.1 µg/ml), polyclonal antibodies against phosphor-histone H3^Ser10 (Santa Cruz Biotechnology; 0.1 µg/ml), BUBR1 (Bethyl Laboratories, Montgomery, TX, USA; 0.2 µg/ml), MAD2 [43 (link)], and p31^comet [44 (link)]. Antibodies against cyclin B1 (1 µg/ml) were gifts from Julian Gannon (Cancer Research UK). Immunoprecipitation of MAD2 or CDC20 was performed as previously described [43 (link)]. Antibody against APC4 for immunoprecipitation was prepared by immunizing rabbits with an APC4 peptide (CIVIKVEKLDPELDS) (GenScript, Piscataway, NJ, USA).

The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam. The CREST antibody was purchased from Antibodies Incorporated. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma for Western blotting. The fluorophore attached secondary antibodies for immunofluorescence analyses were purchased from Jackson Immunoresearch.

Cellular proteins were extracted followed by immunoprecipitation overnight at 4 °C with mouse anti-cdc20 monoclonal antibody (Epitomics, Burlingame, CA, USA) and A/G-agarose beads. The precipitated beads were washed with ice-cold cell lysis buffer. The immune complex was subjected to SDS-PAGE gel electrophoresis followed by immunoblotting assay using rabbit anti-BubR1, Mad2 (Bethyl Laboratories, Inc., Montgomery, TX, USA), and cdc23 antibodies (Epitomics).